Characterization of G12 Sandwich ELISA, a Next-Generation Immunoassay for Gluten Toxicity

2012 ◽  
Vol 95 (2) ◽  
pp. 372-376 ◽  
Author(s):  
Elisabeth Halbmayr-Jech ◽  
Elisabeth Hammer ◽  
Richard Fielder ◽  
Jacqueline Coutts ◽  
Adrian Rogers ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Celiac Disease ◽  
Working Group ◽  
Sandwich Elisa ◽  
Extraction Methods ◽  
Cross Reactivity ◽  
Next Generation ◽  
Preliminary Results ◽  
Sample Extraction

Abstract In this work, a monoclonal antibody called G12, raised against the most immunotoxic peptide to celiac disease patients, was used to develop a sandwich ELISA. Preliminary results on cross-reactivities, recoveries, and extraction methods of the new assay are presented. The assay calibration was performed using material from the Prolamin Working Group. The antibody's specificity was determined by cross-reactivity studies on different grains, nuts, oils, and starches. Recovery of the assay was determined by spiking experiments on common food matrixes, as well as on problematic matrixes. Furthermore, sample extraction methods using ethanol, cocktail solution, and a proprietary buffer have been compared.

Characterization of a monoclonal antibody (HB-22) and development of an ELISA for human apolipoprotein A-I

1995 ◽  
Vol 41 (8) ◽  
pp. 1150-1158 ◽  
Author(s):  
T C Rothwell ◽  
V S Kamanna ◽  
F Y Jin ◽  
E Koren ◽  
T Foley ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Binding Capacity ◽  
Cross Reactivity ◽  
High Density Lipoproteins ◽  
Tween 20 ◽  
Major Protein ◽  
Binding Studies ◽  
Very Low Density Lipoproteins ◽  
Human Apolipoprotein

Abstract We describe the production and characterization of a high-affinity monoclonal antibody, HB-22, for apolipoprotein (apo) A-I, a major protein of human high-density lipoproteins (HDL). Including Tween 20 in the reaction mixture increased the binding capacity of HB-22 to apo A-I. HB-22 showed monospecific reactivity with HDL or apo A-I, displaying no cross-reactivity with apo A-II, intermediate-, low-, or very-low-density lipoproteins. Immunoaffinity columns with HB-22 (in the absence of Tween 20) showed an immunosorbent capacity of 80 micrograms of apo A-I per milligram of antibody. The immunosorbent capacity of HB-22 for apo A-I was similar in plasma samples from normolipidemic, hypercholesterolemic, or hypertriglyceridemic patients. Comparative binding studies demonstrated that compared with other available monoclonal apo A-I antibodies, HB-22 had the greatest apparent affinity for binding to HDL. A competitive ELISA developed by utilizing HB-22 could detect as little as 20 ng of apo A-I in the reaction mixture. The intra- and interassay CVs of the ELISA were 5.4% and 9.5%, respectively.

Design and preclinical characterization of ALXN1210: A next generation anti-C5 monoclonal antibody with improved pharmacokinetics and duration of action

Immunobiology ◽  
2016 ◽  
Vol 221 (10) ◽  
pp. 1158 ◽  
Author(s):  
Douglas Sheridan ◽  
Zhao-Xue Yu ◽  
Yuchun Zhang ◽  
Rekha Patel ◽  
Melissa Lasaro ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Next Generation ◽  
Duration Of Action

scholarly journals Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced byBacillus cereus

1999 ◽  
Vol 65 (10) ◽  
pp. 4470-4474 ◽  
Author(s):  
R. Dietrich ◽  
C. Fella ◽  
S. Strich ◽  
E. Märtlbauer
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Bacillus Cereus ◽  
Cell Lines ◽  
Cross Reactivity ◽  
Uncharacterized Protein ◽  
Hybridoma Cell Lines ◽  
Different Strains ◽  
Hemolysin Bl

ABSTRACT A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereuswere established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L2 component, and antibody 1C2 was specific for the L1 protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains ofB. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity withB. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.

Isolation and preliminary characterization of a monoclonal antibody that interacts preferentially with the liver isoenzyme of human alkaline phosphatase.

1985 ◽  
Vol 31 (3) ◽  
pp. 381-385 ◽  
Author(s):  
G M Lawson ◽  
J A Katzmann ◽  
T K Kimlinger ◽  
J F O'Brien
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Human Liver ◽  
Clinical Laboratory ◽  
Bone Alkaline Phosphatase ◽  
Cross Reactivity ◽  
Immunological Methods ◽  
Enzymic Assay ◽  
Murine Monoclonal Antibodies

Abstract We have prepared murine monoclonal antibodies against isolated human bone alkaline phosphatase (ALP, EC 3.1.3.1). Hybridoma supernates were separately screened for reactivity against both human liver and bone ALP. Although most antibody-positive hybrids showed similar reactivity against both isoenzymes, one hybridoma produced an antibody that interacted preferentially with liver ALP. This antibody was purified and used to establish an immunoassay to differentiate liver ALP from bone ALP. When equal activities of the two isoenzymes (as determined by a conventional enzymic assay) were measured by the immunoassay, a fivefold greater response was obtained with liver than with bone ALP. The immunoassay can be used to measure the proportions of the bone and liver isoenzymes in mixtures of them. Cross reactivity with human placental and intestinal ALP is less than 3% relative to liver ALP. These findings support the feasibility of developing immunological methods to differentiate these isoenzymes in the clinical laboratory.

Production of a monoclonal antibody specific for ovine immunoglobulin E and its application to monitor serum IgE responses to Haemonchus contortus infection

Parasitology ◽  
1997 ◽  
Vol 114 (4) ◽  
pp. 395-406 ◽  
Author(s):  
F. N. J. KOOYMAN ◽  
P. J. S. VAN KOOTEN ◽  
J. F. HUNTLEY ◽  
A. MacKELLAR ◽  
A. W. C. A. CORNELISSEN ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Haemonchus Contortus ◽  
Immunoglobulin E ◽  
Polyclonal Antibodies ◽  
Sandwich Elisa ◽  
Specific Ige ◽  
Cross Reactivity ◽  
Total Serum ◽  
Western Blots ◽  
Serum Ige

Part of the Cε3–Cε4 region of the ovine immunoglobulin E (IgE) gene (nucleotides 1111–1575) was amplified by PCR. The recombinant protein (recIgE1-2) was expressed in E. coli and both monoclonal and polyclonal antibodies were produced. These antibodies recognized recIgE1-2 and native IgE on Western blots and in ELISA. The polyclonal serum showed cross-reactivity with other sheep immunoglobulin classes. The monoclonal antibody was specific for ovine IgE and goat IgE. Infection of sheep with the abomasal nematode Haemonchus contortus resulted in elevated IgE levels in serum 2–4 weeks after infection, as measured by sandwich ELISA using the rabbit polyclonal as capture antibody and the monoclonal antibody against ovine IgE as second antibody. A negative correlation between worm counts and total serum IgE levels at the end of the experiment was found in repeatedly infected sheep. Significant increased levels of excretory–secretory antigens specific IgE levels were found after H. contortus infection. In contrast, no significant changes in 3rd-stage larvae (L3) antigen-specific IgE titre in sera could be detected after infection.

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